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STUDIES     OF     HEMOLYTIC     STAPHYLOCOCCI 

HEMOLYTIC  ACTIVITY — BIOCHEMICAL  REACTIONS — SEROLOGIC  REACTIONS 


LOUIS      A.      J  U  L  I  A  N  E  L  L  E 


Reprinted  from  THE  JOURNAL  OF  INFECTIOUS  DISEASES 
Vol.  31.  No.  3.  September,  1922,  pp.  256-284 


STUDIES  IN  HEMOLYTIC  STAPHYLOCOCCI 

HEMOLYTIC  ACTIVITY— BIOCHEMICAL  REACTIONS  — 
SEROLOGIC  REACTIONS 


A  Thesis  Submitted  to  the  Graduate  School  of  the  University  of 
Pennsylvania  in  Partial  Requirement  for  the  Degree  for  the  Doctor  of 
Philosophy. 


LOUIS    A.    JULIANELLE 


57: 


o 

BIOLOGY 

LIBRARY 

6 


STUDIES    OF    HEMOLYTIC    STAPHYLOCOCCI 

HEMOLYTIC  ACTIVITY BIOCHEMICAL  REACTIONS — SEROLOGIC  REACTIONS 

LOUIS      A.      JULIANELLE 

From  the  Bacteriological  Laboratories  of  the   School  of  Hygiene,    University   of  Pennsylvania 
and  the  Philadelphia  General  Hospital,  Philadelphia 

I.     STUDY    OF    HEMOLYTIC    ACTIVITY 

That  staphylococci  lake  blood  was  brought  out  in  1900,  when  Kraus  *  noticed 
the  hemolytic  effect  of  staphylococci  on  bloodplates.  The  following  year  Neisser 
and  Wechsberg2  demonstrated  a  hemolytic  substance  in  nitrates  of  broth  cul- 
tures. They  found  that  in  alkaline  beef  broth,  this  hemolytic  substance  began 
to  appear  on  the  fourth  day  and  reached  a  maximum  between  the  eighth  and 
fourteenth  day.  In  a  general  way  they  showed  that  aureus  and  virulent 
strains  produced  greater  quantities  of  hemolysins  than  did  either  the  albus 
or  avirulent  strains.  Van  durme 3  found  that  the  hemolytic  power  was  gen- 
erally greatest  in  cultures  freshly  isolated  from  pathologic  conditions,  and 
was  generally  absent  in  cultures  from  dust  and  from  the  normal  mouth.  Todd,4 
working  with  B.  megatherium  and  Kraus B  working  with  staphylococcus  showed 
that  this  action  takes  place  in  vivo  as  well  as  in  vitro. 

PRODUCTION     OF     HEMOLYSIN 

It  had  been  observed  that  in  a  general  way  staphylococci  would  show 
hemolysis  to  a  greater  or  less  extent  on  blood-agar  plates  within  24 
hours.  In  addition,  the  hemolysis  was  not  typical  of  an  exogenous 
hemolysin,  as  is  typical  of  Streptococcus  hemolyticus;  but  rather 
resembled  an  exogenous  product  of  metabolism,  as  in  the  case  of  B.  coli, 
where  the  hemolysis  diffuses  haphazardly  through  the  medium. 

The  first  experiment  was  made  to  determine  what  analogy  there  was 
in  chronicity  in  the  production  of  hemolysins  on  blood  plates  and  in 
broth.  It  might  be  stated  here  that  all  the  work  on  hemolytic  activity  was 
obtained  with  4  cultures  representative  of  all  the  strains  studied.  Two 
were  known  hemolytic,  and  2  were  originally  isolated  as  nonhemolytic. 
Twenty-four  hour  cultures  were  seeded  into  10%  horse  (inactivated) 
serum  broth  in  Erlenmeyer  flasks  and  incubated  at  37  C.  for  24  hours. 
At  the  end  of  each  24-hour  period,  5  c  c  of  the  culture  were  removed 

Received  for  publication,  May  22,  1922. 

1  Wien.  Klin.  Wchnschr.,  1900,   13,  p.  49. 

2  Ztschr.  f.   Hyg.   u.   Infektionskr.,   1901,   36,  p.   299. 

3  Hyg.  Rundschau,  1903,  13,  p.  66. 

4  Trans.  London  Path.  Soc.,  1902,  53,  p.  196. 
B  Wien.   klin.  Wchnschr.,    1902,   15,  p.   382. 


494486 


4  L.    A.    JULIANELLE 

aseptically  and  centrifuged  at  high  speed  for  5  minutes.  One  c  c  of 
the  clear  supernatant  fluid  was  added  to  1  c  c  of  a  washed  2.5%  horse- 
blood  suspension  and  incubated  at  37  C.  for  2  hours,  at  the  end  of 
which  time  the  tubes  were  read  for  hemolysis.  The  concentration  of 
blood  attempted  to  approximate  as  closely  as  possible  the  conditions  of 
the  blood  plate. 

It  was  found  that  no  estimable  hemolysins  were  produced  in  broth 
cultures  within  24  hours.  In  fact,  as  will  be  borne  out  later,  no 
hemolysins  were  shown  to  be  present  until  the  sixth  day.  It  may  be  that 
the  discrepancy  in  time  between  plate  and  broth  cultures  is  explainable 
on  the  grounds  that  in  the  former  case  the  hemolysins  are  so  concen- 
trated around  each  colony  as  to  assert  themselves  at  a  conspicuously 
earlier  period ;  whereas  in  the  latter  case  the  hemolysins  go  into  solution 
and  become  too  dilute  to  have  any  effect  on  a  suspension  of  blood  cells. 

The  next  experiment  was  planned  to  obtain  the  curve  for  the  produc- 
tion of  hemolysins.  The  technic  employed  was  the  same  as  in  the 
preceding  experiment,  except  for  one  detail.  The  cultures  were  seeded 
into  tubes  containing  10  c  c  of  the  serum  broth,  and  at  the  end  of  each 
day  one  tube  was  removed  from  the  incubator  and  used  for  the  tests. 
Care  was  taken  to  keep  the  volume  of  the  tubes  constant  by  adding 
sterile  salt  solution  to  repair  any  loss  by  evaporation. 

Table  1  shows  that  hemolysins  begin  to  appear  on  the  sixth  day, 
reach  a  maximum  at  the  ninth  and  tenth  days,  and  disappear  between 
the  thirteenth  and  sixteenth  days. 

With  the  period  of  hemolysin  production  established,  the  logical 
sequence  was  to  determine  if  possible  the  source  or  the  cause  of  the 
production.  It  was  assumed  entirely  theoretically  that  hemolysis  is 
caused  by  one  of  the  following  or  perhaps  combination  of  factors : 

1 .  Reaction :    An  increase  or  decrease  in  hydrogen-ion  concentration 
sufficient  to  cause  hemolysis. 

2.  Tonicity :    An  increase  or  decrease  in  the  tonicity  of  the  medium 
sufficient  to  cause  crenation  or  laking  of  the  blood  corpuscles. 

3.  Hemotoxin :     A  hemolytic  substance  elaborated  and  secreted  by 
the  bacterial  cell,  causing  hemolysis. 

4.  Proteolysis:     The  production  by  the  bacterial  cell  of  some  sub- 
stance for  the  utilization  of  the  blood  protein.    Under  this  head  would 
be  included  autolytic  products  also. 

In  order  to  establish  experimentally  which  hypothesis  was  correct 
the  following  procedure  was  adopted  :  Coincidental  with  testing  for  the 


HEMOLYTIC  STAPHYLOCOCCI 


presence  of  hemolysins,  the  hydrogen-ion  concentration  was  read  on  the 
Clark  and  Lubs  6  scale ;  the  amino  acidity  was  titrated  by  the  Sorensen  7 
method  ;  the  proteose  content  was  determined  by  the  Vernon  tests ; 8  and 
numerical  counts  made  at  the  end  of  each  day,  as  long  as  was  deemed 
necessary  for  the  points  at  hand. 

TABLE    i 

PRODUCTION    OF    HEMOI.YSINS 


D 

ays 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 

16 

Strain  Al 
(From  Air) 
Hemolysis  

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

o 

0 

54 

W 

56 

W 

56 

54 

"18 

58 

50 

50 

50 

50 

4  6 

38 

36 

36 

Proteose  content  
H-ion  concentration.  . 

0.25 
7.9 

0.25 
7.9 

0.25 
8.0 

0.25 
8.1 

0.25 
8.2 

0.3 
8.3 

0.3 
8.3 

0.3 
8.3 

0.3 

8.3 

0.3 
8.3 

0.3 
8.3 

0.3 
8.3 

0.3 

8.4 

0.3 

8.4 

0.3 
8.4 

0.3 

8.4 

Strain  A5 
(From  Air) 
Hemolysis  

0 

0 

0 

0 

0 

0 

-4- 

+ 

+  +  + 

+  -f 

+1 

+ 

+ 

-f 

•+• 

0 

Amino  acidity  

**> 

04 

64 

v> 

84 

80 

98 

102 

88 

84 

84 

84 

T> 

72 

44 

44 

Proteose  content  
H-ion  concentration.  . 

0.25 
7.7 

0.25 
7.9 

0.25 
8.0 

0.25 
8.0 

0.3 
8.2 

0.3 
8.3 

0.35 
8.4 

0.4 
8.4 

0.4 
8.4 

0.4 
8.4 

0.4 
8.4 

0.46 

8.4 

0.45 
8.4 

0.45 
8.4 

0.45 
8.4 

0.45 
8.4 

Strain  H2 
(From  Heart  Blood) 
Hemolysis  

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

0 

o 

0 

Amino  acidity  

54 

56 

56 

56 

56 

56 

54 

52 

46 

44 

44 

44 

38 

36 

34 

34 

Proteose  content  
H-ion  concentration.  . 

0.25 
7.7 

0.25 
7.9 

0.25 
8.0 

0.25 
8.2 

0.25 
8.2 

0.3 
8.2 

0.3 
8.2 

0.3 
8  2. 

0.25 
8.2 

0.3 
8.3 

0.3 
8.3 

0.3 
8.3 

0.3 
8.4 

0.3 

8.4 

0.3 

8.4 

0.3 
8.4 

Strain  T9 
(From  Throat) 
Hemolysis  

0 

0 

0 

0 

0 

0 

•+- 

•-  + 

+  + 

+ 

+ 

4. 

-f- 

0 

o 

o 

Amino  acidity  

54 

W 

56 

56 

56 

54 

86 

74 

66 

56 

56 

56 

58 

44 

?R 

38 

Proteose  content  
H-ion  concentration.  . 

0.25 

7.7 

0.25 
7.9 

0.25 
8.0 

0.25 
8.1 

0.25 
8.2 

0.3 
8.2 

0.3 
8.2 

0.3 
8.2 

0.3 
8.3 

0.3 
8.3 

0.3 
8.3 

0.35 
8.3 

0.35 
8.4 

0.35 
8.4 

0.35 
8.4 

0.35 

8.4 

Control. 


Hemolyis    Amino  Acidity 
0  38 


Proteose 
0.25 


H-ion 
7.5 


Figures  for  proteose  content  represents  amount  of  medium  required  to  equal  1  c  c  ol 
standard. 

Figures  for  amino  acidity  represent  c  c  of  20/N  NaOH  required  to  neutralize  100  c  c  of 
medium. 

Plus  signs  indicate:   +,  25%  hemolysis;    ++,  50%;   +  ++,  75%. 

Bacterial  Counts  Made  With  Production  ol  Hemolysin  Stains 


Al 

A5 

H2 

T9 

0  

40,000 

72,000 

50,000 

73,000 

24  

100.000.000 

180,000,000 

450,000,000 

300,000,000 

48  

830,000,000 

ooo.G(y>,ooo 

850,000,000 

1,000,000,000 

72  

1,210,000,000 

6.300.000,000 

8,900,000,000 

9,200,000,000 

96  

7,000.000.000 

1,000.000,000 

2,500,000,000 

2,000,000,000 

120  

460,000,000 

300,000,000 

3,200,000  000 

800,000,000 

144  

150,000,000 

350,000,000 

800,000,000 

600,000000 

1.  An  analysis  of  the  results   (table  1  and  chart  1)  shows  several 
points :    The  ultimate  reaction  of  all  the  cultures — hemolytic  and  non- 

•  Jour.  Bacteriol.,   1917,  2,  p.    109. 

7  Biochem.  Ztschr.,   1908,   7,  p.  45. 

8  Jour.  Physiol.,  1904,  30,  p.  330. 


6  L.    A.    JULIANELLE 

hemolytic  alike — is  the  same,  PH  8.4.  If  the  hemolysis  were  the  effect 
of  reaction,  all  the  cultures  should  show  a  like  behavior  on  blood.  But 
since  the  cultures  do  not  show  the  same  hemolytic  activity,  it  is  reason- 
able to  exclude  reaction  as  the  cause  of  hemolysis.  Incidentally,  sterile 
salt  solution,  the  reaction  of  which  is  adjusted  to  PH  8.4,  does  not  cause 
hemolysis. 

Charts  1   and  2. — Showing  counts,  amounts  of  amino  acids   and  hemolytic 
substances  produced. 


12     13       14       IB 


Fig.   2. — Strain  AS. 


2.  No  effort  was  made  to  determine  the  tonicity  of  the  cultures. 
The  impression  was  gathered  from  the  work  of  Larson  et  al.9  that 
bacteria  of  themselves  do  not  change  the  surface  tension  of  mediums, 
and  in  their  study  specific  depressants  were  added  when  a  drop  in 
surface  tension  was  desired. 

•  Larson,  Cantwell,  and  Hartzell:  Jour.  Infect.  Dis.,   1919,  25,  p.  41. 


HEMOLYTIC  STAPHYLOCOCCI  7 

3.  The   figures   for  the  numerical   counts   show   that  there  is.  an 
increase  in  the  number  of  staphylococci  until  the  third  day,  when  a 
maximum  is   reached.     From  then  on  there  is  a  sharp  decrease  in 
numbers,  indicating  that  growth  of  an  active  nature  at  least  has  come 
to  a  cessation.     If  the  production  of  hemolysins  and  the  numerical 
counts  had  shown  a  parallelism,  it  could  have  been  reasonably  assumed 
that  the  hemolysin  were  a  true  secretion  product  and  a  definite  hemo- 
toxin.     Since  they  show  no  such  parallelism,  however,  the  hemolysin 
must  be  of  some  other  nature. 

4.  The  course  of   proteolysis   or  amine  acidity  runs  a  definitely 
parallel  course  to  the  curve  of  hemolysin  production.     The  suggestion 
offered  itself  that  if  not  directly  associated,  then  some  close  relationship 
must  exist  between  the  two.    Further  study  reveals  the  following  con- 
catenation of  events:   (1)  the  period  of  maximum  growth  occurs  on 
the  third  and   fourth  day;    (2)   the  maximum  production  of  amino 
acidity  occurs  on  the  seventh  and  eighth  days;  (3)  the  maximum  pro- 
duction of  hemolysins  occurs  on  the  ninth  and  tenth  days.     Stated  in 
another  way,  the  growth  period  precedes  the  amino  acidity  period,  which 
in  turn  precedes  the  hemolysin  production  period.    It  would  seem  from 
such  an  interrelated  process  that  the  production  of  hemolysins  is  a 
proteolytic  process  and  perhaps  even  autolytic. 

There  is  one  other  point  of  interest  brought  out  by  this  experiment. 
Although  there  is  an  increase  in  amino  acidity,  there  is  no  corresponding 
decrease  in  proteose  content.  This  is  probably  due  to  the  fact  that  the 
biuret  test,  used  in  determining  the  amount  of  proteose  present,  shows 
the  presence  of  substances  other  than  proteose;  so  that  even  if  proteose 
were  proteolyzed  to  form  polypeptides,  peptides  and  the  higher  amino 
acids  the  intensity  of  the  color  would  still  remain  the  same.  One  other 
point — it  shows  that  the  production  of  erepsin  by  staphylococci  enables 
them  to  attack  peptones  and  proteoses. 

5.  Following  the  suggestion  offered  in  the  foregoing  experiment, 
the  next  step  was  to  determine  what  role  autolysis  plays  in  hemolysis. 
For  this  purpose  24-hour  cultures  were  inoculated  in  Erlenmeyer  flasks 
(10%  serum  broth),  and  incubated  at  37  C.  for  5  days.    This  culture 
was  then  distributed  in  equal  volumes  into  test  tubes.     To  one  series 
was  added  0.25%  phenol,  to  a  second  10%  HCC13;  a  third  series  was 
incubated  at  45  C. ;  and  a  fourth  was  left  untreated  and  incubated  with 
the  first  and  second  series  at  37  C.    The  object  of  this  procedure  was 
to  determine  whether  after  the  maximum  growth  period  was  reached 


8 


L.    A.    JULIANELLE 


and  the  cultures  were  inactivated  by  chemical  or  heat,  with  the  enzymes 
still  capable  of  activity,  hemolytic  substances  were  being  produced.  Each 
day  tests  were  made  for  the  presence  of  hemolysins.  After  the  first 
day,  guinea-pig  serum  and  a  living  (24-hour)  culture  in  1  c  c  quantities 
were  added  to  the  45  C.  specimen.  This  was  to  supply  complement,  if 
it  were  needed,  and  any  other  vital  substances  necessary  for  hemolysis 
that  a  growing  culture  might  possess.  The  results  are  appended  in 
table  2,  which  shows:  1.  No  hemolysin  was  formed  in  cultures  sub- 
jected to  antiseptics  or  heat.  2.  Complement  does  not  appear  necessary 
for  hemolysis.  3.  A  living  culture  produces  hemolysis  per  se,  and  is 

TABLE     2 

SHOWING    EFFECT    OF    HEAT    AND    CHEMICAL    AGENTS    ON    PRODUCTION    OF    HEMOLYTIC 

SUBSTANCE 


Da 

ys 

1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

Strain  A5  (From  Air) 
Phenol  

HCCls  

4  



45C  

__ 





45  C.  +  complement  

* 



45  C.  +  24  hour  culture  

* 

4- 

4- 

4- 

+ 

4- 

+ 

4- 

* 

* 

Untreated  

+ 

+ 

4- 

+ 

+ 

+ 

4- 

-t- 

Phenol  

HCCls  

45  C  

45  C.  +  complement  

* 

4- 

45  C.  +  24  hour  culture  

•  * 

4- 

4- 

4- 

4- 

+ 

-f 

4- 

* 

it 

Untreated  

4- 

4- 

+ 

4- 

4- 

+ 

+ 

-(- 

Phenol  

HCCls  

Complement  

* 

Strain  A5  

» 

+ 

4- 

i 

4- 

4. 

+ 

+ 

* 

* 

Strain  T9  

* 

4- 

+ 

+ 

4- 

+ 

4- 

+ 

* 

* 

Salt  

*  =:  no  test  conducted.     Day  1  is  1st  day  under  treatment,  but  6th  day  of  age  of  culture. 

consequently  worthless  in  such  an  examination.  These  results  do  not 
show  that  hemolysin  is  of  an  autolytic  nature ;  neither  do  they  show 
that  it  is  not  of  an  autolytic  nature.  The  conclusion  to  be  drawn  is 
that  in  the  case  of  heat,  the  hemolysin  being  thermolabile,  is  possibly 
dissipated  ;  while  in  the  case  of  the  antiseptics,  the  hemolysin  is  so  closely 
associated  with  the  bacterial  cell  that  destruction  of  the  latter  means  lack 
of  manifesation  from  the  former.  This  falls  somewhat  in  line  with  the 
work  of  Gordon  on  meningocbcci,  showing  that  hemolysins  are  endocel- 
lular  and  are  liberated  on  autolysis  of  the  bacterial  cells. 

Effect   on   Hemolytic   Activity   of   Successive    Transplantation    in 
Blood-Free   Medium. — The    object    of   the   next    experiment   was   to 


HEMOLYTIC  STAPHYLOCOCCI  9 

determine  whether  a  hemolytic  strain  of  staphylococcus  is  always 
hemolytic.  No  definite  references  to  the  loss  of  this  haemolytic 
manifestation  could  be  found  in  the  literature.  Transplants  were 
made  daily  into  peptone  broth,  and  at  the  end  of  each  week  blood- 
agar  plates  were  streaked  to  show  whether  the  cultures  were  still 
hemolytic.  After  the  second  week,  since  the  plates  were  readily 
hemolyzed,  the  cultures  were  transplanted  every  other  day,  and  after 
the  first  month  every  week.  The  reason  for  this  change  of  procedure 
was  the  assumption  that  by  daily  transplantations  the  cultures  were  kept 
very  active  and  that  it  would  be  more  difficult,  if  possible,  to  suppress  so 
vital  a  quality. 

This  experiment  was  continued  for  more  than  four  months,  and  at 
the  time  of  writing  the  cultures  were  still  hemolytic.  On  some  occasions 
there  appeared  to  be  retardation  in  hemolysins,  and  then  the  following 
week  the  cultures  were  as  actively  hemolytic  as  originally.  -Since  the 
retardation  was  neither  progressive  nor  continuous,  it  is  reasonable  to 
assume  that  it  was  probably  due  to  differences  in  the  blood  used  for  the 
work.  Normal  horse  blood,  which  was  used  for  the  blood-agar  plates 
in  this  experiment,  has  been  shown  by  Neisser  and  Wechsberg 2  to 
possess  small  quantities  of  antihemolysin.  This  normal  quantity,  how- 
ever, may  have  been  sufficient  to  delay  nemolysis.  It  would  seem, 
therefore,  that  hemolytic  cultures  tend  to  remain  hemolytic. 

Effect  on  Nonhemolytic  Strains  of  Successive  Transplantations  in 
Blood  Medium. — In  this  case  the  point  at  hand  was  to  determine 
whether  nonhemolytic  cultures  could  be  made  hemolytic  by  adaptation 
to  blood  medium.  If  nonhemolytic  cultures  can  be  made  to  lake  blood, 
it  may  be  said  that  any  strain  of  staphylococcus  is  hemolytic,  adding 
provisionally  that  continual  adaptation  to  a  blood-free  habitat  ultimately 
suppresses  its  hemolytic  activity  and  keeps  it  in  abeyance ;  but  readapta- 
tion  to  a  blood-containing  medium  will  restore  the  suppressed  activity. 
Table  3  shows  that  after  a  period  of  7  months,  certain  strains  regained 
their  hemolytic  ability.  It  may  be  that  this  power  was  recovered  at  an 
earlier  period,  but  tests  were  not  definitely  made  until  the  stated  lapse 
of  time. 

It  should  be  added  here,  in  view  of  a  wealth  of  work  in  a  hospital 
laboratory,  that  we  think  every  strain  of  staphylococcus  is  definitely 
hemolytic.  The  strains  will  vary  in  degree  of  hemolysis,  and  in  rapidity 
of  hemolysis,  but  if  sufficient  time  is  given,  all  strains  will  show  hemol- 
,  ysis.  When  the  strains  under  study  were  isolated,  a  period  of  6  days 
was  given  to  determine  hemolysis  on  blood  plates,  and  it  is  now  apparent 


10 


L.    A.    JULIANELLE 


that  the  6  days  were  not  sufficient.  Other  cultures  not  included  in  this 
survey  did  show  hemolysis  after  the  arbitrarily  chosen  time,  and 
attempts  to  collect  nonhemolytic  strains  after  10  and  in  rarer  cases  12 
days,  have  failed.  So  that  it  seems  by  virtue  of  this  evidence  that 
the  strains  we  originally  labeled  nonhemolytic  were  in  reality  hemolytic, 
and  that  their  hemolytic  character  was  very  much  suppressed.  It 
would  seem,  therefore,  that  it  can  be  definitely  stated  that  cultures 
which  did  not  show  hemolysis  within  6  days  were  able  to  give  definite 
signs  of  hemolysis  on  blood  plates  within  24  hours  and  complete 
hemolysis  within  48  hours. 

Since  the  completion  of  this  experiment  every  strain  of  staphylo- 
coccus  isolated  (whether  a  contaminant  or  a  pathogen)  was  held  for 
study.  The  number  of  days  required  to  show  beginning  hemolysis  was 
recorded.  These  results  are  tabulated  in  table  4.  It  will  be  seen  that 
every  strain  shows  hemolysis,  but  that  the  factor  of  time  plays  an 

TABLE     3 
DEVELOPMENT  OF  HEMOLYSIS  BY  NONHEMOLYTIC  ?  STRAINS 


April, 

November, 

April, 

November, 

1921 

1921 

1921 

1921 

Al 

No  hemolysis 

Hemolysis 

P5 

i    No  hemolysis 

Hemolysis 

A2 

Hemolysis 

Hemolysis 

S  2 

Hemolysis 

Hemolysis 

A3 

Hemolysis 

Hemolysis 

Tl 

No  hemolysis 

Hemolysis 

A5 

Hemolysis 

Hemolysis 

T2 

Hemolysis 

Hemolysis 

Fl 

No  hemolysis 

Hemolysis 

T3 

Hemolysis 

Hemolysis 

H2 

No  hemolysis 

Hemolysis 

T5 

Hemolysis 

Hemolysis 

PI 

Hemolysis 

Hemolysis 

T6 

Hemolysis 

Hemolysis 

P2 

Hemolysis 

Hemolysis 

T8 

No  hemolysis 

Hemolysis 

P3 

Hemolysis 

Hemolysis 

T9 

Hemolysis 

Hemolysis 

P4 

Hemolysis 

Hemolysis 

X 

No  hemolysis 

Hemolysis 

important  part.  Thus  it  is  seen  that  in  a  general  way  aureus  strains 
show  hemolysis  earlier,  and  that  virulent  strains  also  show  hemolysis 
earlier  than  the  saprophytic ;  but  the  point  is  clear  that  white  and  aureus 
strains,  saprophytic  and  parasitic  alike,  become  hemolytic.  In  the  case  of 
nipples,  for  example :  These  are  supposedly  sterilized  and  sent  to  the 
laboratory  to  be  tested  for  sterility  so  that  it  is  logical  to  assume  that  any 
growth  is  apt  to  be  contamination.  Yet  the  9  white  strains  are  as 
rapidly  hemolytic  as  the  18  orange  strains  isolated  from  pus. 

Effect   of   Carbohydrates   on   Hemolysis. — It  has  been   shown  by 
Ruediger,10   Lyall,11   Davis,12   Sekiguchi,13    Stevens   and   Koser 14   and 

">  Ibid.,  1906,  3,  p.  663. 

«  Jour.  Med.  Res.,  1914,  30,  p.  SIS. 

«  Davis:  Jour.  Infect.  Dis.,  1917,  21,  p.  308. 

"  Ibid.,  1917,  21,  p.  475. 

14  Jour.  Exper.  Med.,  1919,  30,  p.  539. 


HEMOLYTIC  STAPHYLOCOCCI 


11 


others,  that  carbohydrates  prevent  hemolysis  by  streptococcus,  and  it 
was  problematic  just  what  their  effect  on  staphylococcus  hemolysis 
would  be.  Two  experiments  were  carried  out  to  determine  this  point. 
In  the  one  case,  cultures  were  planted  into  10%  serum  broth  plus  1% 
dextrose.  After  9  days  tests  were  made  for  hemolysis  and  H-ion 
concentration  read — to  assure  ourselves  that  an  acidity  would  not  inter- 
fere with  the  test.  The  results  were:  for  10%  serum  broth,  dextrose 
1%,  strain  A5  gave  PH  6.4,  hemolysis  and  strain  T9,  PH  7.6  and 
hemolysis. 

Incidentally  both  these  strains  were  streaked  on  lactose-blood-agar 
plates,  and  in  both  cases  hemolysis  was  produced  within  24  hours.  In 
the  second  case,  cultures  were  planted  into  peptone  broth  plus  \% 
dextrose.  The  test  for  hemolysis  was  positive  after  24  hours,  but  the 

TABLE     4 
HEMOLYTIC  ACTIVITY  OF  CONSECUTIVE  CULTURES 


Source 

Pigment 

No.  of 

Cultures 

Average  Time  for 
Hemolysis 

Air  

White 

8 

6-7  days 

Sputum  

Yellow 

White 

2 
1 

1-2  days 
3  days 

Skin  

Aureus 
White 

2 
4 

1  day 
4  days 

Throat  

White 

4 

1  day 

Tonsil  .         

Yellow 
White 

1 
1 

1  day 
1  day 

Nipple  

Aureus 
White 

1 
9 

1  day 
1-2  days 

Feces  

Aureus 
Yellow 

White 

2 
2 

2 

1  day 
1  day 
3  days 

Pus  

Yellow 
Yellow 

1 
3 

2  days 
2  days 

Necropsy  

Orange 
Aureus 

18 
3 

1-2  days 
1-2  days 

Water  

Yellow 
White 

1 

2 

1  day 
4  days 

Contamination  (source  unknown  — 

White 

3 

1  days 

hemolysis  was  not  typical,  showing  a  browning  similar  to  acid  hematin 
formation.  Consequently  the  PH  value  was  determined  and  found  to 
be  4.4.  The  reaction  was  adjusted  to  neutrality  and  hemolysis  no  longer 
took  place.  Approaching  the  question  from  another  tangent,  sterile 
salt  solution  adjusted  to  a  reaction  of  PH  4.4  caused  the  same  type  of 
hemolysis. 

It  would  seem  from  these  experiments  that  carbohydrates  do  not 
influence  hemolysis  as  produced  by  staphylococcus.  Regarding  the 
acidity  produced  in  the  peptone  broth  and  not  in  the  serum  broth,  it  is 
easily  conceivable  that  the  buffer  qualities  of  the  serum  in  the  latter 
obscure  the  acid  formed  by  fermentation  of  dextrose. 


12  L.    A.    JULIANELLE 

Effect  of  Heat  on  Hemolysis. — Neisser  and  Wechsberg  2  found  that 
heating  the  staphylococcus  "hemolysin"  for  20  minutes  at  56  C.  would 
completely  inactivate  it. 

In  determining  the  effect  of  heat  on  the  hemolytic  action,  the 
supernatant  fluids  of  centrifuged  9-day  cultures  were  heated  at  56  C. 
for  30  minutes,  and  it  was  found  that  the  hemolysin  of  staphylococcus 
is  a  thermolabile  substance,  which  can  be  destroyed  by  heating  in 
this  way. 

DISCUSSION 

Previous  investigators  of  the  hemolytic  activity  of  staphylococcus 
were  concerned  with  observations  of  the  hemolytic  activity  per  se.  Aside 
from  some  speculations  as  to  its  relation  to  pigment,  virulence  and 
agglutination,  no  attempt  was  made  to  arrive  at  its  causation.  The 
point  under  study  here  was  concentrated  on  the  cause  of  the  hemolytic 
activity,  and  the  period  of  its  development  was  only  a  coincidental 
observation,  since  this  phase  of  it  was  already  sufficiently  elaborated  by 
previous  investigators. 

Our  results  point  to  a  process  of  proteolysis — perhaps  associated 
with  autolysis — -as  the  cause  of  hemolysis.  This  is  not  a  new  conception 
— it  has  been  shown  to  be  the  fundamental  of  meningococcus  hemolysis 
and  were  experiments  performed  to  establish  the  point  of  possibly  B. 
proteus,  B.  coli,  etc.  Although  we  have  been  unable  to  demonstrate 
irrevocably  that  autolysis  is  the  specific  cause,  it  is  very  significant  that 
the  period  of  maximum  growth  first  appears,  then  the  period  of 
maximum  amino  acidity,  and,  finally,  the  period  of  maximum  hemolysis. 
Such  a  sequence  of  evidence  can  point  only  to  autolysis. 

It  must  be  for  this  reason  that  we  have  been  unable  to  suppress  the 
hemolytic  activity  of  our  hemolytic  strains.  If  hemolysis  is  due  to  so 
important  a  function  as  protein-splitting,  the  factor  involved  is  too 
vital  to  be  eradicated  by  continued  growth  in  blood-free  mediums. 
Conversely,  it  is  no  wonder  that  slowly  hemolytic  cultures  will  increase 
in  rapidity  of  hemolysis  by  continued  adaptation  to  an  environment 
where  protein  ultilization  becomes  more  pronounced. 

Nor  is  it  phenomenal  that  sugar  should  not  inhibit  hemolysis  in 
such  a  case.  Kendall  and  Walker's  15  conception  that  the  presence  of 
glucose  has  a  protein  sparing  effect  and  consequently  retards  production 
of  proteolytic  enzymes  can  be  accepted  only  provided  the  hydrogen-ion 
concentration  of  the  medium  increases  within  suitable  limits.  For  as 

15  Jour.  Infect.  Dis.,   1915,  17,  p.  442. 


HEMOLYTIC  STAPHYLOCOCCI  13 

Berman  and  Rettger 16  pointed  out,  in  tests  in  which  buffers  are 
employed  proteolytic  enzymes  appear  as  soon  in  sugar  mediums  as  in 
plain  broth.  And  in  serum  broth,  the  buffer  qualities  of  serum  cannot 
be  denied. 

IT.    RELATIONSHIP    OF    HEMOLYTIC    ACTIVITY    TO    OTHER 
METABOLIC    ACTIVITIES 

This  part  of  the  investigation  concerns  itself  with  a  study  of  the 
biochemical  reactions  of  the  staphylococci,  particularly  as  possible  rela- 
tions to  hemolysis.  Although,  as  the  evidence  submitted  will  show, 
hemolysis  appears  to  be  a  separate  entity  from  the  biochemical  reactions 
pursued,  some  new  points  of  interest  have  been  added  to  the  literature 
of  the  hemolytic  staphylococci. 

CHROMOGENESIS 

Except  in  a  general  way,  a  distinction  of  the  chromogenic  varieties 
of  the  staphylococci  is  an  insignificant  one.  The  pigment  produced  by 
bacteria  is  influenced  to  a  greater  or  less  extent  by  the  medium  employed 
for  its  production,  and  can  be  greatly  modified  by  selection  or  by 
previous  environment.  Loeffler's  serum  medium,  for  example,  without 
affecting  the  inherent  power  of  chromogenesis  always  accentuates  the 
depth  of  color  produced  by  staphylococci.  Pigment  will  vary  with  the 
amount  of  oxygen,  the  amount  of  moisture  available,  and  the  age  of  the 
culture. 

So  Neisser  and  Lipstein17  offer  the  hypothesis  that  white  cocci  were  origi- 
nally orange  cocci  which  have  lost  their  chromogenic  power.  Rodet  and  Cour- 
mont 18  published  the  observation  of  the  transformation  of  a  white  staphylo- 
coccus  to  an  aureus  and  subsequently  to  a  white  again.  Lubinski 19  showed  that 
the  orange  forms  lost  their  pigment  when  grown  anaerobically;  in  some  cases 
the  recovery  was  delayed  and  in  other  cases  the  loss  was  permanent.  Kolle 
and  Otto 20  stated  that  chromogenic  cocci  lose  their  chromogenesis  by  heating 
to  85  C.,  by  prolonged  cultivation  on  artificial  mediums,  and  by  repeated  ani- 
mal passage.  Winslow  and  Rogers 21  showed  that  a  temperature  of  50-55  C. 
may  cause  a  loss  in  chromogenesis. 

Neisser  and  Wechsberg  showed  that  strains  of  both  Staphylococcus  albus 
and  Staphylococcus  aureus  would  produce  hemolysins.  This  was  later  cor- 
roborated by  both  Kutscher  and  Konrich22  and  Koch.23  Noguchi24  and  Rosen- 

16  Jour.  Bact.,  1918,  3,  p.  389. 

17  Handbuch.  d.  pathog.   Mikroorganismen,    1914,  3,  p.   105. 

18  Compt.  rend.  Acad.  d.  sc.,  1890,  9,  p.   186. 
18  Centralbl.   f.   Bakteriq^.,    1894,    16,  p.   769. 

20  Ztschr.  f.   Hyg.  u.  Infektionskr.,   1902,  41,  p.   369. 

21  Jour.  Infect.  tDis.,   1906,  3,  p.  485. 

22  Zetschr.  f.   Hyg.   u.   Infektionskr.,    1904,   48,   p.   249. 
»  Ibid.,  1907,  58,  p.  287. 

24  Arch.  f.  klin.   Chir.,  1911,  96,  p.  696. 


14  L.     A.     JULIANELLE 

bach  **  show  a  relation  between  virulence  and  pigmented  cocci,  while  Passet 2b 
and  Fisher  and  Levy27  show  that  the  lightly  colored  or  colorless  forms  are 
most  often  associated  with  disease  processes. 

EXPERIMENTS 

In  determining  chromogenesis  the  technic  employed  was  that  sug- 
gested by  Winslow  and  Winslow.28  Cultures  were  grown  on  agar 
slants  at  20  C.  for  2  weeks.  A  portion  of  the  growth  was  spread  over 
white  roughened  paper,  with  a  platinum  loop  and  allowed  to  dry  in  air. 
The  hue  and  tint  were  matched  against  the  colors  of  the  frontispiece 
of  their  book  (table  5). 

TABLE     5 
SOURCES  AND   CHROMOGENESIS   OF  THE   STRAINS    STUDIED 

A  1—  From  the    air Lemon  yellow  I 

A  2 — From  the   air Medium  cadmium  yellow  IV 

A  3— From  the    air Cadmium  orange  III 

A  5 — From  the    air Medium  cadmium  yellow  IV 

F  1— From  feces   White 

H2 — From  heart's  blood  at  necropsy Medium  cadmium  yellow  V 

PI — From  pus  from   spine Cadmium  orange  IV 

P  2 — From  pus   from  carbuncle Lemon  yellow  I 

P  3 — From  pus  from   acne Cadmium  orange  IV 

P4 — From  pus  from  extracted  tonsil Cadmium  orange  IV 

P  5— From  pus  (unclassified)  Cadmium  orange  IV 

S  2— From  skin    White 

T  1 — From  throat    Orange  yellow  III 

T  2 — From  throat    Medium  cadmium  yellow  V 

T  3 — From  throat    Medium  cadmium  yellow  VI 

T  5— From  throat    Lemon  yellow  II 

T  6— From  throat    Lemon  yellow  III 

T  8— From  throat    White 

T  9— From  throat    Orange  yellow  V 

X— From  blood  culture   (case  ferunculous) Orange  yellow  III 

C15— From  throat    White 

C16— From  throat    White 

CIS— From  throat    White 

J  1— From  pus    White 

L  1— From  pus    Cadmium  orange  IV 

It  will  be  seen  at  a  glance  that  there  is  no  relationship  between  pig- 
ment and  hemolysis.  The  cultures  are  all  hemolytic,  and  yet  they  vary 
from  a  white  to  a  rich  golden  brown.  This  is  scarcely  surprising.  The 
literature  shows  that  pigment  production  may  be  varied,  and  while 
the  hemolytic  activity  seems  to  be  fixed,  it  could  hardly  be  expected  that 
the  two  functions  would  be  related. 

ACID    PRODUCTION    IN    THE    PEPTONE    MEDIUM    OF    CLARK    AND    LUBS 

Preparatory  to  the  carbohydrate  metabolism  studies  of  staphylococci, 

this  experiment  was  made  to  determine  in  a  general  way  any  relation- 

• 

25  Dent.  med.  Wchnschr.,  1884,  6,  p.  31. 

28  Passet:  Fortschr.   d.  Med.,  1885,   33,  p.   33. 

»  Dent.  Ztschr.  f.  Chir.,  1893,  36,  p.  94. 

28  Systematic  Relationships  of  the  Coccocese,   1908. 


HEMOLYTIC  STAPHYLOCOCCI 


15 


ship  between  hemolysis  and  acid  production.  In  view  of  the  methyl 
red  test  of  differentiation  of  B.  coli  and  B.  aerogenes  by  this  medium, 
it  seemed  at  the  time  that  it  might  possess  some  value  in  this  work. 
The  peptone  medium  contained  0.5%  K2HPO4,  0.5  peptone  (Difco), 
and  0.5%  dextrose,  and  was  adjusted  to  PH  7.4. 

Table  6  shows  the  H-ion  readings  of  the  different  cultures  from  time 
to  time  as  specified.  With  the  exception  of  Al,  all  strains  reach  an 
end-point  of  PH  4.2-4.6  within  96  hours.  Although  there  seem  to  be 
differences  in  the  earlier  readings,  there  is  no  line  of  demarcation 
between  the  acid  production  of  the  cultures.  These  differences  are 
probably  explainable  on  differences  in  numbers  inoculated,  periods  of 
lag,  etc. 

TABLE     6 

ACID    PRODUCTION    IN    CLARK    AND    LUBS    MEDIUM  * 


8 
Hours 

12 
Hours 

16 
Hours 

20 
Hours 

24 
Hours 

48 
Hours 

72 
Hours 

96 
Hours 

A  1.... 

7.6 

76 

7.4 

7.0 

69 

69 

69 

6  9 

A  2  

6.4 

4.6 

4.6 

4.6 

4.6 

4  4 

4.4 

4.4 

A3  

6"> 

4  6 

4  4 

4  4 

4  4 

44 

4  4 

4  4 

A  5  

4  6 

4  4 

4  2 

4  2 

4  9 

4  2 

4  2 

4  ' 

F  1  

6.0 

5  8 

5  8 

50 

46 

4  6 

46 

4  6 

H2  

5.0 

4  6 

4.4 

4  4 

4  4 

4  4 

4  4 

4  4 

P  1  

6.1 

4.9 

4  6 

4  6 

4.6 

4.6 

4  6 

46 

P  2  

6.0 

4.6 

4.6 

4.6 

4.6 

4.6 

4.6 

4.6 

P  3  

68 

4  6 

4  6 

4  6 

4  6 

4  6 

4  6 

4  6 

P  4  

5.0 

50 

48 

4  6 

4  6 

4  6 

4  6 

4  6 

P  5.  ... 

6.6 

5.0 

4  6 

4  4 

4.4 

4.4 

4  4 

4  4 

S   2  

7.6 

5.8 

5.0 

5.0 

4.6 

4.6 

4  8 

4  6 

T  1  

5.0 

5.0 

4.8 

4.8 

4.6 

46 

4.6 

4.6 

T  2  

5.4 

4.8 

4.8 

-4.6 

4.6 

4.6 

4.6 

4.6 

T  3  

50 

4.9 

4  9 

4  9 

4  6 

4  4 

4  4 

4  4 

T  5  

5.0 

48 

4.6 

4  4 

4.4 

4.4 

4.4 

4  4 

T  6  

7.6 

6.6 

6.0 

5.4 

4.6 

4.6 

4.6 

4.6 

T  8  

5.5 

5.0 

5.0 

4.8 

4.8 

48 

4.6 

4.6 

T  9  

7.4 

5  6 

5.0 

50 

4  6 

4  6 

4  6 

4.6 

X  

6.4 

4.4 

4.4 

4.4 

4.4 

4.4 

4.4 

4  4 

Control  

7.2 

7.2 

7.2 

7.2 

7.2 

7.2 

7.2 

7.2 

*  Figures  represent  -values  of   H-ion   concentration. 

At  this  point,  the  question  arose  as  to  what  determined  the  acid 
end-point  of  the  cultures.  To  approach  an  answer,  2  experiments  were 
planned :  ( 1 )  Cultures  were  grown  in  the  same  medium  with  the  reac- 
tion adjusted  to  PH  4.4;  (2)  cultures  which  had  already  reached  an 
acidity  of  PH  4.4  were  killed  by  heating  at  56  C.  for  30  minutes  and 
inoculated  with  a  24-hour  culture. 

In  both  these  cases,  the  H-ion  concentration  'was  increased  to  4.2 
and  4  after  24  hours.  It  might  be  of  interest  to  quote  here  the  work 
of  Hall  and  Frazer  29  who  found  that  staphylococci  could  reach  a  H-ion 
concentration  of  2.6 — an  end-point  which  exhibited  no  relation  to  sapro- 
phytic  or  pathogenic  forms. 


29  Abstract,  Lancet,   1921,   18,  p.   912. 


16  L.    A.    JULIANELLE 

CARBOHYDRATE     METABOLISM 

In  view  of  the  diagnostic  importance  of  the  fermentative  reaction  of  the 
colon-typhoid  group,  it  was  deemed  advisable  to  devote  considerable  attention 
to  this  subject.  Very  little  previous  work  has  been  done  on  the  ability  of  the 
staphylococci  to  ferment  carbohydrate  mediums.  Of  course,  it  is  common 
knowledge  that  they  attack  the  more  familiar  sugars  with  the  production  of 
acid,  but  no  gas.  Gordon,30  in  reporting  a  classification  study  of  the  white 
cocci,  gave  the  fermentation  reactions  on  lactose,  maltose,  glycerol  and  man- 
nitol.  Dudgeon 31  reported  a  comparative  study  of  the  aureus  and  albus  cocci, 
studying  among  other  things  their  acid  production  in  11  carbohydrate  mediums; 
but  none  of  his  results  were  quantitative.  Winslow  and  Winslow 2S  studied 
glucose  and  lactose,  and  Kligler  32  glucose,  lactose  and  sucrose.  More  recently 
Winslow  and  his  co-workers  M  made  a  quantitative  study  of  the  acid  produced 
in  9  different  sugars.  They  found  more  than  half  the  strains  studied  fermented 
glucose,  maltose  and  sucrose ;  about  half  fermented  lactose ;  5  strains  fermented 
salicin,  1  strain  each  fermented  inulin  and  raffinose,  and  no  strains  fermented 
dulcitol  and  mannitol. 

In  our  study,  we  have  employed  17  carbohydrates  in  all-dextrose, 
galactose,  levulose,  sucrose,  lactose,  maltose,  raffinose,  arabinose,  inulin, 
dextrin,  salicin,  adonitol,  mannitol,  sorbitol,  dulcitol,  glycerol  and  starch. 
Twenty- four  hour  cultures  were  inoculated  into  1%  peptone  broth  plus 
1%  of  the  carbohydrate  designated.  The  cultures  were  incubated  at 
37  C.  for  one  week,  and  the  PH  value  determined  by  matching  the 
tubes  against  the  Clark  and  Lubs  6  standards.  In  table  7  the  PH  values 
alone  are  given,  since  gas  was  not  formed  in  any  case. 

The  table  shows  that  the  carbohydrates  are  either  fermented  or  not ; 
but  in  either  case  the  reaction  is  uniform.  There  are  slight  differences 
in  some  of  the  mediums,  but  they  are  not  important  enough  for 
classification ;  they  indicate  merely  functional  differences  and  as  such 
are  negligible. 

To  compress  the  table : 

Carbohydrates  Fermented  Not  Fermented 

Glucose  Starch 

Galactose  Dulcitol 

Levulose  Adonitol 

Sucrose  Dextrin 

Lactose  Inulin 

Maltose  Arabinose 

Salicin  Raffinose 
Mannitol 
Sorbitol  ' 
Glycerol 

30  Quoted   by    Winslow    and    Winslow.      Supplement   to    the    34th   annual    report    of    local 
gov't.  bd.  containing  the  report  of  the  Med.  officer  for   1904-1905,  p.   387. 
81  Jour.  Path.  &  Bacteriol.,  1908,   12,  p.  242. 
32  Jour.  Infect.   Dis.,   1913,   12,  p.  432. 
38  Winslow,  Rothberg  and  Parsons:    Jour.  Bacteriol.,   1920,  5,  p.   145. 


HEMOLYTIC  STAPHYLOCOCCI 


17 


The  discrepancy  in  uniformity  of  fermentation  between  this  study 
and  that  of  Winslow  and  others  is  possibly  due  to  the  fact  that  they 
included  in  their  survey  strains  of  Staph.  epidermidis,  ureae,  candidus, 
tetragenus,  candicans,  aureus  and  aurianticus,  thereby  making  a  survey 
of  many  less  active  organisms  than  those  employed  in  our  study. 

PROTEIN     METABOLISM 

Decomposition  of  Peptone  to  Amino  Acids. — As  a  rule,  the  only 
accessible  figures  of  amino  acid  formation  of  staphylococci  occur 
scattered  through  bacteriologic  literature  where  the  question  at  hand 
was  primarily  a  study  of  the  nitrogen  metabolism  of  several  species  and 

TABLE     7 
FERMENTATION    OF    CARBOHYDRATES 


Arabinose 

Dextrose 

Galactose 

Levulose 

Sucrose 

Lactose 

Maltose 

a 

"3 

a 

Dextrin 

Adonite 

Salicin 

Mannitol 

Sorbitol 

Ducitol 

Glycerol 

i 

o 

"v            ° 
«          1 

£       tf 

A  1. 

7.4 

'6.2 

5.9 

7.0 

6.2 

6.2 

6.2 

7.8 

7.1 

7.6 

6.0 

6.2 

6.0 

7.7 

6.0 

8.0  —  7.8 

A  2 

7.3 

4.4 

4.7 

5.0 

5.0 

5.0 

4.8 

7.5 

7.1 

7.6 

6.0 

4.6 

4.7 

7.7 

6.0 

8.0  —  7.7 

A  3 

7.2 

4.6 

4.7 

5.0 

4.8 

5.6 

4.8 

7.5 

7.1 

7.6 

6.0 

5.0 

4.7 

7.7 

6.0 

8.0  —  7.6 

A  5 

7.2 

4.4 

4.6 

5.0 

4.8 

5.0 

4.8 

7.5 

7.6 

7.6 

5.8 

5.0 

4.7 

7.7 

6.0 

8.0  —  7.8 

F  1 

7.0 

4.6 

4.6 

5.0 

4.9 

5.0 

4.8 

7.5 

7.8 

7.6 

6.0 

5.4 

4.7 

7.7 

6.0 

8.0  —  7.7 

H2 

7.3 

4.4 

4.7 

5.0 

4.8 

5.0 

4.8 

7.5 

7.1 

7.6 

6.0 

5.2 

4.7 

7.8 

6.0 

8.0  —  7.7 

P  1 

7.3 

4.4 

4.7 

5.0 

4.6 

5.1 

4.6 

7.5 

7.1 

7.6 

6.0 

5.0 

4.7 

7.7 

6.0 

8.0  —  7.7 

P  2 

7.2 

4.4 

6.0 

5.2 

4.8 

5.0 

4.8 

7.5 

7.1 

7.5 

5.4 

5.0 

4.7 

7.7 

6.0 

8.0  —  7.8 

P  3 

7.3 

4.5 

4.7 

5.2 

4.8 

5.0 

6.0 

7.5 

7.1 

7.6 

6.0 

4.6 

4.7 

7.7 

5.0 

8.0  —  7.8 

P  4 

7.2 

4.4 

4.7 

5.0 

4.7 

5.1 

5.4 

7.5 

7.2 

7.6 

6.0 

4.6 

4.7 

7.6 

5.0 

8.0  —  7.8 

P  5 

7.3 

4.9 

5.1 

5.2 

4.9 

5.1 

5.6 

7  r 

7.1 

7.6 

6.2 

5.4 

4.7 

7.7 

6.0 

8.0  —  7.5 

S  2 

7.3 

4.6 

4.7 

5.2 

4.8 

5.4 

4.8 

7~.5 

7.T 

7.6 

6.0 

4.6 

4.7 

7.7 

6.0 

8.0  —  7.7 

T  1 

7.3 

4.4 

4.7 

5.0 

4.8 

5.0 

4.8 

7.5 

7.1 

7.8 

6.0 

5.3 

4.7 

7.6 

5.2 

8.0  —  7.7 

T  2 

7.3 

4.4 

4.7 

5.0 

4.6 

5.1 

4.8 

7.5 

7.2 

7.6 

6.0 

5.2 

4.7 

7.7 

6.0 

8.0  —  7.7 

T  3 

7.3 

4.4 

4.7 

5.0 

5.0 

5.0 

4.2 

7.6 

.7.2 

7.6 

-6.2 

4.6 

4.7 

7.7 

5.0 

8.0  —  7.7 

T  5 

7.2 

4.4 

4.7 

5.0 

4.8 

5.1 

4.8 

7.3 

7.2 

7.6 

6.0 

4.6 

4.7 

7.7 

6.0 

8.0  —  7.7 

T  6 

7.3 

4.6 

4.7 

5.2 

4.8 

5.0 

4.8 

7.5 

7.1 

7.6 

6.0 

4.6 

4.7 

7.7 

6.0 

8.0  —  7.7 

T  8 

7.3 

4.4 

4.8 

5.0 

4.8 

5.6 

4.8 

7.5 

7.0 

7.4 

6.0 

4.6 

4.7 

7.6 

5.0 

8.0  —  7.6 

T  9 

7.3 

4.6 

4.9 

5.0 

4.8 

5.2 

4.8 

7.5 

7.1 

7.4 

6.0 

5.2 

4.7 

7.7 

6.0 

8.0  —  7.9 

X 

7.3 

4.4 

4.7 

5.2 

5.0 

5.0 

4.8 

7.5 

7.1 

7.4 

5.4 

5.4 

4.7 

7.0 

5.0 

8.0  —  7.7 

Control 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0 

7.0  —  7.2 

one  or  two  strains  of  staphylococci  were  fortuitously  included.  Our 
object  here  was  to  give  a  definite  conception  of  the  amino  acid  digestion 
of  peptone,  and  incidentally  to  use  such  an  expedient  for  a  classification, 
if  possible. 

Rosenthal  and  Patai8*  found  that  the  curve  of  amino  acid  production  by 
staphylococcus  underwent  an  initial  sharp  rise  within  24  hours,  and  this  was 
followed  by  a  more  gradual  rate  of  increase  until  the  fifth  and  sixth  day.  Also 
virulent  organisms  produced  more  amino  acid  than  the  avirulent  ones.  Their 
determinations  were  made  by  the  S^rensen  method.  The  work  of  Berman  and 
Rettger M  shows  that  at  the  end  of  1  week  3  strains  of  aureus  reached  an 

at  Centralbl.  f.  Bakteriol.,  I,  O.,  1914,  73,  p.  406. 
86  Jour.  Bacteriol.,  1918,  3,  p.  367. 


18 


L.    A.    JULIANELLE 


amino  acid  figure  equivalent  to  47  c  c  of  20/NaOH,  and  one  strain  of  albus,  :i 
figure  of  52  c  c  of  20/NaOH.    They  also  used  the  S0rensen  method. 

Benton  38  recently  observed  that  in  1.5%  peptone  broth,  staphylococcus  shows 
a  decrease  in  amino  acidity  until  the  5th  day,  with  a  following  rise  until  the 
7th  day;  in  2%  peptone  broth,  the  decrease  continues  until  the  3rd  day  with  a 
gradual  increase  until  the  9th  day;  in  pure  ascitic  fluid,  after  a  1  day  decrease, 
there  is  a  rise  until  the  4th  day.  She  used  the  Van  Slyke  method  for  amino 
acid  determination. 

In  our  own  experiment,  a  2%  Difco  peptone  extract  broth  was 
employed.  The  tubes  were  inoculated  with  a  24-hour  growth  and  at  the 
end  of  each  day  the  amino  acidity  was  determined  by  the  SpYensen 
method  (table  10). 

TABLE     8 
AMINO  ACID   DECOMPOSITION   OF  PEPTONE  * 


1st  Day 

2d  Day 

3d  Day 

4th  Day 

5th  Day 

PH 

A.  A. 

PH 

A.  A. 

PH 

A.  A. 

PH 

A.  A. 

PH 

A.  A. 

A  1... 

7.5 
7.5 
7.5 
7.5 
7.5 
7.5 
7.8 
7.7 
7.5 
7.5 
7.3 
7.7 
7.8 
7.5 
7.5 
7.5 
7.5 
7.7 
7.6 
7.8 
7.0 

40.0 
60.0 
68.0 
80.0 
68.0 
68.0 
50.0 
56.0 
56.0 
56.0 
52.0 
48.0 
80.0 
56.0 
56.0 
56.0 
56.0 
56.0 
44.0 
52.0 
48.0 

7.5 
7.7 
7.7 
7.5 
7.5 
7.5 
7.9 
7.7 
7.9 
7.9 
7.4 
7.8 
7.9 
7.9 
7.9 
7.6 
7.8 
7.9 
7.7 
7.8 
7.0 

48.0 
48.0 
68.0 
72.0 
72.0 
96.0 
56.0 
80.0 
84.0 
72.0 
68.0 
76.0 
116.0 
84.0 
72.0 
72.0 
68.0 
116.0 
80.0 
72.0 
48.0 

7.5 
7.6 
7.7 
7.6 
7.8 
7.7 
7.8 
7.7 
8.0 
7.9 
7.5 
7.8 
7.9 
7.9 
7.9 
7.8 
7.7 
8.0 
7.8 
7.8 
7.0 

72.0 
84.0 
88.0 
92.0 
96.0 
112.0 
96.0 
96.0 
96.0 
100.0 
76.0 
72.0 
116.8 
96.0 
96.0 
88.0 
92.0 
116.0 
100.0 
92.0 
48.0 

7.5 
7.7 
7.7 
7.6 
7.8 
7.8 
7.S 
7.7 
7.7 
7.7 
7.5 
7.8 
8.0 
7.9 
7.9 
7.5 
7.7 
7.7 
7.8 
7.8 
7.0 

100.0 
112.0 
116.0 
120.0 
120.0 
116.0 
116.0 
116.0 
116.0 
92.0 
76.0 
92.0 
140.0 
80.0 
96.0 
80.0 
72.0 
132.0 
72.0 
116.0 
48.0 

7.5 
7.9 
7.9 
8.0 
8.0 
8.0 
8.0 
8.0 
7.7 
7.7 
8.0 
8.0 
8.0 
8.0 
8.0 
8.0 
7.9 
7.7 
7.9 
8.0 
7.0 

44.0 
72.0 
79.0 
76.0 
60.0 
72.0 
79.0 
72.0 
64.0 
68.0 
64.0 
£0.0 
68.0 
68.0 
68.0 
40.0 
600 
104.0 
64.0 
64.0 
48.0 

A  2  

A  3  

A  5  

PI  

H2  

PI  

P  2     

P  3  

P  4  

P  5  

S  2  

T  1  

T  2  

T  3  

T  5  

T  6  

T  8  

T  9  

X  

Control  

*  In  PH  column,  Pn  readings  are  given.      In  A.  A.  column,  figures  represent  number  of  c  c 
of  20/N  NaOH  required  to  neutraliz  100  c  c  of  culture. 

The  significant  features  brought  out  are  that  at  the  end  of  the  first 
day  there  is  either  a  slight  increase  or  decrease  in  amino  acid,  then  a 
gradual  rise  to  the  4th  day,  with  a  falling  off  on  the  5th  day.  At  the 
time,  we  assumed  that  a  maximum  had  been  reached  on  the  4th  day. 
This  checks  fairly  well  with  the  results  of  Rosenthal  and  Patai,  but 
brings  our  maximum  a  bit  sooner  than  was  the  case  in  Benton's  work. 

These  results  differ  materially  from  those  obtained  with  serum 
broth,  but  the  mediums  were  of  course  different.  It  would  seem  that 
in  serum  broth  the  amino  acids  are  simultaneously  formed  and  utilized, 


38  Jour.  Infect.  Dis.,  1919,  25,  p.  231. 


HEMOLYTIC  STAPHYLOCOCCI 


19 


and  thus  the  figures  are  kept  low ;  whereas  in  peptone  broth,  the  amino- 
acidity  figures  increase  rapidly  due  to  the  greater  amounts  of  peptone 
present. 

Production  of  Ammonia. — This  test  was  performed  for  a  double 
purpose :  In  the  first  place,  it  was  interesting  to  determine  what 
happened  to  the  amino  acid  formed,  and  in  the  second  place,  to 
determine  whether  any  differentiation  could  be  made  on  this  basis.  The 
amount  of  ammonia  formed  was  measured  daily  for  5  days  after  incu- 
bating at  37  C.  The  medium  employed  was  composed  of  \%  peptone 
and  0.05%  K2HPO4.  The  tubes  were  sealed  with  paraffin  to  prevent 
the  escape  of  ammonia.  The  determination  was  made  with  Nessler 
reagent,  and  the  cultures  were  matched  against  a  known  standard  by 

TABLE     9 
AMMONIA    FORMATION 


2d  Day 

3d  Day 

4th  Day 

5th  Day 

A  1... 

6.4 

9.72 

11  60 

21  84 

H2  

13.80 

19.20 

20  40 

14  64 

H3  

12.00 

17.76 

20.50 

1644 

H5  

12.00 

15.36 

23.40 

1692 

F  1  

960 

20.40 

24  00 

26  04 

H2  

14.16 

21.84 

2704 

21  00 

PI  

12.84 

14.60 

16.80 

1678 

P  2  

12.60 

14.60 

19.20 

21  84 

P  3  

9.60 

21.60 

30.00 

21  84 

P  4  

17.16 

20.40 

30.00 

32.88 

P  5  

9.60 

14.16 

15  48 

3000 

S   2  

12.96 

14.40 

2820 

2400 

T  1  

24.00 

27.00 

48.00 

4800 

T  2  

13.32 

30.00- 

32.88 

21.36 

T  3  

16.78 

23.20 

24.00 

14.64 

T  5  

14.76 

21.60 

32.80 

28  ?0 

T  6  

14.16 

17.96 

32.80 

28  56 

T  8  

32.80 

38.40 

64.80 

57.12 

T  9  

14.60 

16.68 

32.80 

14.60 

X  

19.20 

23.40 

24.00 

16  44 

Control  

1.98 

1.98 

1.98 

1.98 

Figures  represent  mg.  of  NHs  as  nitrogen  per  100  c  c  of  culture. 

means  of  the  Dubosq  colorimeter.  From  table  9  it  will  be  seen  (1)  that 
all  the  cultures  produce  ammonia,  and  (2)  that  amino-acidity  and 
ammonia  formation  are  simultaneous  processes.  Winslow,  Rothberg 
and  Parsons  report  positive  ammonia  formation  in  all  but  11  strains 
out  of  180  studied. 

Reduction  of  Nitrates.  —  Gordon  30  and  Winslow,28  Rothberg  and  Parsons  M 
found  that  nitrate  reduction  by  staphylococci  was  a  more  or  less  general  char- 
acter. Winslow  and  Winslow28  reported  only  21%  aureus  and  13%  albus 
reducers.  Kligler's  study  showed  7  out  of  11  aureus  and  only  1  out  of  12 
albus  reduced  nitrates.  The  more  recent  work  of  Winslow,  Rothberg  and 
Parsons w  had  the  advantage  of  better  technic  and  should  have  the  greatest 
weight. 


20  L.    A.    JULIANELLE 

In  making  our  determination,  the  medium  contained  1%  peptone, 
0.5%  K2HPO4  and  \%  KNO3.  The  cultures  were  incubated  for  one 
week  at  37  C,  and  the  presence  of  nitrates  was  determined  by  the 
sulphanilic  acid — a — naphthalamine  method.  All  the  strains  except  A 
were  able  to  reduce  nitrates. 

Formation  of  Indol. — In  a  survey  of  the  literature  of  indol  production  by 
staphylococci,  3  references  have  been  found  of  a  positive  nature.  Emmerling38 
described  the  production  of  indol  afer  14  days'  cultivation  under  anaerobic 
conditions  on  an  egg  white  medium.  Tissier  and  Martelly*9  reported  positive 
indol  by  a  culture  of  Staphylococcus  albus  isolated  from  meat,  and  cultivated 
in  a  fibrin  medium.  Distaso4"  isolated  an  atypical  Staphylococcus  which  was 
an  obligate  anaerobe  and  showed  inability  to  attack  any  sugar,  but  which  was 
capable  of  forming  indol.  The  results  of  the  first  two  are  questionable  on 
account  of  the  technic  employed,  while  the  third  case  is  concerned  with  an 
atypical  organism.  On  the  other  hand,  negative  indol  production  is  reported 
by  Buard,41  Seltzer,42  Dobrowski,48  Distaso,44  Zipfel,45  Herzfeld  and  Klinger,46 
Winslow,  Rothberg,  and  Parsons,33  and  Bayne-Jones  and  Zimmiger.47 

Our  tests  were  made  by  cultivating  in  a  medium  of  \%  peptone  and 
0.5%  K2HPO4  at  37  C.  Tests  for  the  presence  of  indol  were  made 
on  the  first,  third,  fifth,  seventh  and  tenth  day  after  incubation  by  the 
para-dimethyl-amido-benzald-benzaldehyde  method.  All  tests  were 
negative. 

Action  on  Milk. — Table  10  gives  the  reaction  of  each  strain  in 
litmus  milk.  It  will  be  seen  that  after  10  days'  incubation  at  37  C., 
one  strain  shows  no  apparent  change  in  reaction,  11  strains  show  acid 
production,  and  8  strains  show  acid  with  coagulation  and  liquefaction. 
The  PH  values  in  lactose  broth  has  been  placed  alongside  the  milk  reac- 
tions. As  was  expected,  the  reaction  coincides. 

Liquefaction  of  Gelatin. — In  determining  gelatin  liquefaction,  an 
effort  was  made  toward  a  quantitative  study.  The  technic  employed 
was  to  inoculate  gelatin  tubes  with  0.1  c  c  of  a  24-hour  broth  culture 
(diluted  if  necessary  to  insure  an  even  turbidity).  The  amount  of 
gelatin  liquefied  was  measured  by  determining  the  number  of  c  c  from 
a  mark  drawn  at  the  original  level  of  the  gelatin  to  the  level  of  the 

88  Berlin  der  Deutsch.   Chem.   Gessellsch.,   1896,  29,  p.   27.21. 

89  Ann.   de  1'Inst.   Pasteur.,   1910,   24,  p.   865. 

40  Centralbl.  f.   Bakteriol.,   I,   O.,   1912,   62,  p.  433. 

41  Compt.  rend.   Soc.  de  biol.,  1908,   65,  p.   158. 

42  Centralbl.  f.  Bakteriol.,  I,   O.,    1909,  51,  p.   465. 

43  Ann.  de  1'Inst.  Pasteur.,   1910,   24,  p.   595. 

44  Centralbl.  f.  Bakteriol.,  I,  O.,   1911,   59,  p.   10? 

46  Ibid.,    1913,   67,   p.   572. 
4B  Ibid.,    1915,  76,  p.   1. 

47  Bull.    Johns    Hopkins    Hosp.,    1921,    32,    p.    299. 


HEMOLYTIC  STAPHYLOCOCCI 


21 


nonliquefied  gelatin.  The  cultures  were  incubated  at  20  C.  for  21  days 
unless  the  gelatin  was  entirely  liquefied  before  that  time,  when  the 
liquefaction  was  estimated. 


TABLE    10 

SHOWING    ACTION    ON    MILK 


1  Day 

3  Days 

5  Days 

7  Days 

10  Days 

Lactose 

A  1  

No  change 

No  change 

No  change 

No  change 

No  change 

6.2 

A  2  

Acid 

Acid 

Acid 

Acid 

Acid 

5.0 

A  3 

Coagulation 

Coagulation 

Liquefaction 

5.6 

A  5 

Acid 

Coagulation 

Coagulation 

Liquefaction 

5.0 

P  1 

Acid 

Coagulation 

Coagulation 

Liquefaction 

5.0 

H  2 

Liquefaction 

5.0 

PI  

No  change 

Acid 

Acid 

Acid 

Acid 

5.1 

P  2  

No  change 

No  change 

Acid 

Acid 

Acid 

5.0 

P  3  

No  change 

Acid 

Acid 

Coagulation 

Liquefaction 

5.0 

P  4  

Acid 

Acid 

Acid 

Acid 

Acid 

5.1 

P  5  

Acid 

Acid 

Acid 

Acid 

Acid 

5.1 

8  2  

No  change 

No  change 

No  change 

Acid 

Acid 

5.4 

T  1  

No  change 

Acid 

Acid 

Coagulation 

Liquefaction 

5.0 

T  2  

No  change 

No  change 

No  change 

Acid 

Coagulation 

5.1 

T  3  

No  change 

Acid 

Acid 

Acid 

Acid 

5.0 

T  5  

No  change 

No  change 

No  change 

No  change 

No  change 

5.1 

T  6  

No  change 

No  change 

No  change 

Acid 

Acid 

5.0 

T  8  

Acid 

Acid 

Acid 

Acid 

Acid 

5.6 

T  9  

Acid 

Acid 

Acid 

Acid 

Coagulation 

5.2 

X  

Acid 

Coagulation 

Acid 

5.0 

TABLE     11 
LIQUEFACTION    OF   GELATIN 


Amount 
Liquefied 

No.  of 
Days 

A  1... 

No  liquefaction 

21 

A  2.... 

5.2  c  c 

19 

A3  

No  liquefaction 

21 

A  5  

7.1  cc 

19 

Fl  

No  liquefaction 

21 

H2  

No  liquefaction 

21 

PI  

6.0  c  c 

19 

P  2  

1.0  c  c 

21 

P3  

5.5  c  c 

18 

P  4  

No  liquefaction 

21 

P5  

No  liquefaction 

21 

82  

3.0  cc 

19 

T  1  

6.7  cc 

17 

T  2  

5.5  C  C 

17 

T3  

0.9  C  c 

21 

T  5  

3.8  cc 

19 

T  6  

No  liquefaction 

21 

T  8  

No  liquefaction 

21 

T  9  

4.1  c  c 

21 

X.       .                                                              

2.0  c  c 

21 

Control  

No  liquefaction 

21 

Manner  of  Liquefaction :  Some  time  later  the  study  of  gelatin 
liquefaction  was  extended  by  an  observation  of  the  manner  of  lique- 
faction. Table  12  gives  a  graphic  representation  of  the  findings.  Up 
to  the  10th  day,  let  us  say,  there  is  a  pseudodifferentiation  of  2  types : 
one  type  giving  a  saucer-shaped  liquefaction  and  the  second  type  giving 


22 


L.    A.    JULIANELLE 


a  cone-shaped  liquefaction.  After  that  the  liquefaction  proceeds 
uniformly  in  all  the  cultures  by  stratification.  The  difference,  however, 
is  so  superficial  that  we  would  hardly  suggest  a  classification  on  this 
characteristic. 

One  significant  feature  brought  out  by  the  tables  is  the  ability  of 
six  cultures  to  attack  gelatin-cultures  which  did  not  a  year  previously 
manifest  this  ability.  This  shows  above  all  the  variability  of  the  organ- 
isms to  be  classified — a  variability  which  emphasizes  the  fact  that  in 
order  to  classify  staphylococci  we  must  depend  on  more  substantial 
characters  than  functional  differences. 


TABLE     12 
RESULTS  OF  AGGLUTINATION 


Serums 

Al 

A5 

Pi 

T9 

LI 

A  1  

5120 

0 

20 

40 

20 

A  2  

0 

2560 

1280 

0 

1280 

A3  

0 

2560 

1280 

0 

1280 

A  5  

0 

2560 

1280 

0 

1280 

F  1  

1280 

20 

20 

40 

20 

H2  

80 

320 

320 

0 

1280 

PI  

0 

2560 

1280 

20 

1280 

P  2  

1280 

0 

0 

0 

0 

P  3  

0 

2560 

640 

2560 

1280 

P  4  

80 

640 

320 

0 

640 

P  5  

0 

320 

640 

40 

1280 

S  2  

1280 

0 

0 

0 

0 

T  1  

0 

2560 

640 

0 

1280 

T  2  

640 

2560 

640 

320 

1280 

T  3  .  .. 

0 

2560 

1280 

0 

1280 

T  5  

640 

20 

0 

0 

1280 

T  6  

0 

2560 

1280 

40 

0 

T  8  

640 

0 

0 

20 

40 

T  9  

640 

2560 

1280 

640 

1280 

X  

1280 

2560 

640 

1280 

1280 

C  15  

2560 

160 

80 

80 

20 

C  16  

640 

0 

20 

20 

20 

C  18  

640 

0 

20 

20 

20 

J  1  

80 

1280 

1280 

80 

1280 

L  1  

0 

1280 

1280 

0 

1280 

*  Figures  represent  the  dilution  at  which  agglutination  was  observed  by  naked  eye  read- 
ing.     All  controls  were  negative. 

Reduction  of  Methylene  Blue, — At  the  December,  1921,  meeting  of 
the  Am.  Assn.  of  Bacteriol.,  Avery  reported  his  investigation  of  the 
use  of  methylene  blue  in  differentiating  hemolytic  streptococci  from 
human  and  dairy  sources.  He  found  that  dairy  strains — bovine  and 
cheese — reduced  methylene  blue,  but  that  the  human  strains  did  not. 
Because  of  these  results,  we  tried  reducing  methylene  blue  by  our 
staphylococcus  strains.  The  technic  of  the  test  consisted  in  adding  to  a 
24-hour  broth  culture  varying  dilutions  of  methylene  blue,  and  covering 
with  sterile  paraffin.  The  cultures  were  reincubated  for  a  second  day, 


HEMOLYTIC  STAPHYLOCOCCI  23 

when  the  results  were  read.  It  was  found  that  all  strains  reduced  or 
decolorized  methylene  blue  at  dilutions  of  1 :  50,000  and  1 :  25,000;  they 
showed  partial  decolorization  at  dilution  of  1 :  10,000,  except  strains  T8, 
C15  and  Jl,  which  were  negative;  and  at  a  dilution  of  1 :  1,000  all  the 
strains  were  negative. 

Hydrolysis  of  Sodium  Hippurate. — Ayers  and  Rupp  48  found  that 
hemolytic  bovine  streptococci  could  be  differentiated  from  the  human  by 
the  fact  that  the  former  could  split  sodium  hippurate  into  glycocoll  and 
benzoic  acid.  We  employed  this  test  in  our  study  to  determine  whether 
such  a  procedure  would  be  of  value  in  differentiating  the  staphylococci. 
The  medium  employed  contained  1%  peptone,  1%  sodium  hippurate 
0.015%  K2HPO4,  and  the  reaction  was  adjusted  to  PH  7.2.  The  cul- 
tures were  incubated  at  37  C.  for  7  days.  At  that  time  hydrolysis  was 
determined  by  adding  0.5  c  c  of  a  7  %  FeQ3  solution  for  every  2  c  c  of 
the  culture  medium ;  if  hydrolysis  had  taken  place  an  insoluble 
precipitate  was  formed,  whereas  the  mixture  became  clear  on  standing 
several  minutes  if  hydrolysis  had  not  taken  place.  All  the  cultures  were 
able  to  split  sodium  hippurate. 

RELATION     TO    VIRULENCE 

Although  Neisser  and  Wechsberg 2  showed  that  aureus  and  albus  strains  alike 
are  capable  of  hemolytic  activity,  their  experiment  seems  to  indicate  that 
purely  saprophytic  forms  never  attain  this  faculty.  This  was  corroborated 
later  by  Kutcher  and  Konrich 22  and  also  by  Koch.23  Noguchi  in  presenting 
his  results  stated  that  hemolysis  was  proportional  to  the  virulence  of  a  strain, 
but  the  evidence  he  presents  does  not  justify  such  a  conclusion.  Montegazza  *3 
was  unable  to  demonstrate  any  definite  relation  between  the  intensity  of  an 
infection  and  the  quantity  of  hemolysin  produced. 

In  approaching  an  answer  to  the  question  of  inter-relationship 
between  virulence  and  hemolysis,  two  methods  present  themselves — 
either  hemolytic  strains  will  prove  to  be  virulent,  or  nonhemolytic  strains 
will  be  avirulent. 

Following  the  first  method,  then,  strains  A5,  PI,  P3  and  T9,  all 
definitely  hemolytic,  were  used.  Twenty-four-hour  broth  cultures  of 
each  were  inoculated  in  1  c  c  quantities  into  the  peritoneum  of  separate 
mice.  No  causalties  occurring,  the  mice  were  killed,  the  peritoneums 
were  washed  with  sterile  saline,  and  the  washings  injected  into  a  fresh 
mouse.  Incidentally,  cultures  were  made  of  the  peritoneal  exudate  and 
heart  blood  as  a  check.  This  procedure  was  carried  successively  for 

48  Personal  communication. 

*»  Biochem.  Centralbl.    1908,   8,   p.   226. 


24  L.    A.    JULIANELLE 

3  days  with  3  mice  for  each  strain.    After  the  third  mouse,  in  no  case 
was  staphylococcus  demonstrable  by  smear  or  culture  from  the  peri- 
toneum indicating  complete  overwhelming  of  the  4  strains.     Cultures 
of  the  heart  blood,  which  were  made  to  test  the  invasive  powers  of  the 

4  strains,  were  negative  each  day.     Here,  if  anything,  the  virulence  of 
the  strains  should  have  increased  by  the  animal  passage,  but  instead 
the  organisms  decreased,  the  more  resistant  organisms  lasting  until  the 
third  passage.    This  would  indicate  that  hemolysis  is  quite  independent 
of  virulence. 

Later,  in  attempting  to  isolate  a  virulent  strain,  3  different  strains 
from  pus  were  injected  into  rabbits.  Two  strains  injected  intravenously 
in  amounts  of3ccofa  24-hour  broth  culture  caused  no  apparent  effect. 
The  third  strain 'caused  death  in  0.5  c  c  amounts  within  2  days,  and  0.25 
c  c  amounts  within  1  week,  presenting  in  this  case  typical  staphylococcus 
lesions.  This  strain  was  used  in  our  serologic  work  and  designated  as 
LI.  The  point  of  interest  here,  however,  is  that  although  the  3  strains 
were  distinctly  hemolytic,  only  1  proved  to  be  sufficiently  virulent  to  kill 
a  rabbit.  The  combined  evidence  of  these  7  strains  makes  plausible  the 
conclusion  that  hemolytic  strains  are  not  necessarily  virulent. 

The  second  method — that  nonhemolytic  strains  would  prove  to  be 
avirulent — was  not  tried.  Nonhemolytic  strains  were  not  isolated  during 
the  course  of  the  entire  investigation.  However,  a  glance  at  table  4  at 
this  point  will  show  that  strains  of  an  undoubtedly  saprophytic  character 
are  hemolytic.  In  a  general  way,  perhaps,  the  strains  requiring  the 
greatest  time  for  hemolysis  are  probably  the  least  virulent  of  any ;  but, 
on  the  other  hand,  the  strains  giving  most  rapid  hemolysis  may  be 
saprophytic. 

LEUKOCIDIN     ACTIVITY 

It  was  not  the  purpose  in  this  experiment  to  make  a  study  of  the 
leukocidin  produced  by  staphylococci.  The  subject  has  been  well  worked 
out.  The  purpose  was  rather  to  determine  whether  hemolytic  activity 
bears  any  relation  to  leukocidin  activity. 

Van  de  Velde50  first  demonstrated  leukocidin  by  filtration  in  24-hour  cul- 
tures. Later  he  and  Denys M  showed  that  the  leukocidin  was  not  specific,  but 
was  a  metabolic  product  which  destroyed  other  tissue  cells  as  well  as  leu- 
kocytes. Bail82  obtained  a  maximum  production  of  leukocidin  in  11  days. 
Neisser  and  Wechsberg2  added  considerably  to  the  knowledge  of  staphylo- 
coccus leukocidin.  Making  use  of  the  reduction  of  methylene  blue  by  leuko- 
cytes, they  found  that  leukocidin  appears  in  filtrates  after  4  days  and  reaches 

ro  La  Cellule,  1894,   10,  p.  403. 

51  Ibid.,   1895,   11,  p.  395. 

82  Arch.  f.  Hyg.,  1898,  32,  p.  133. 


HEMOLYTIC  STAPHYLOCOCCI  25 

a  maximum  after  1  week;  that  leukocidin  was  produced  by  white  and  orange 
strains;  that  the  more  virulent  the  strain  the  more  leukocidin  produced;  that 
leukocidin  was  destroyed  by  heating  at  56  C. ;  that  normal  horse  and  immune 
serum  possesses  antileukocidin ;  that  leukocidin  does  not  attack  kidney  cells. 

In  making  our  tests  the  same  strains  used  for  hemolytic  activity  were 
used.  The  cultures  were  inoculated  each  day  into  10%  serum  broth 
for  16  days  so  that  on  the  17th  day  we  had  16  cultures  of  each  strain 
of  from  1  day  to  16  days  old.  The  cultures  were  then  centrifuged  at 
high  speed  for  5  minutes,  and  1  c  c  of  the  supernatant  fluid  was  used 
for  the  test. 

Leukocytes  were  obtained  by  injecting  8-10  c  c  of  sterile  aleuronat 
into  the  pleural  cavity  of  guinea-pigs,  and  after  15  hours  the  animals 
were  bled  to  death  and  the  pleural  exudate  removed  with  a  capillary 
pipet.  An  equal  amount  of  1.5%  sterile  sodium  citrate  was  added  to 
the  cells  to  prevent  coagulation. 

The  presence  of  leukocidin  was  determined  by  the  methylene  blue 
reduction  test.  The  methylene  blue  consisted  of  1  c  c  saturated  solution 
of  methylene  blue,  20  c  c  absolute  alcohol,  and  29  c  c  distilled  water. 
The  minimum  quantity  of  leukocytes  to  reduce  methylene  blue  was 
first  measured  by  using  different  amounts  of  leukocytes  varying  from 
0.2  c  c  to  2  c  c,  the  volume  being  made  equal  through  the  series  with 
sterile  salt  solution.  Two  drops  of  methylene  blue  were  added,  and 
then  the  mixture  was  covered  with  a  layer  of  sterile  liquid  paraffin  to 
prevent  reoxidation  from  the  air.  The  tubes  were  incubated  at  37  C. 
for  2  hours. 

To  twice  the  minimum  quantity  of  the  leukocytes  found  necessary 
to  give  reduction  of  methylene  blue  was  added  1  c  c  of  the  super- 
natant centrifuged  culture.  The  tubes  were  incubated  at  37  C.  for 
11/2  hours,  when  2  drops  of  methylene  blue  and  liquid  paraffin  were 
added.  Incubation  was  continued  for  2  hours  more  when  the  readings 
were  made.  In  case  of  reduction,  no  leukocidins  were  present,  since  the 
leukocytes  had  not  been  injured. 

It  was  found  that  leukocidin  appeared  on  the  4th  day  and  dis- 
appeared on  the  8th  day ;  and  that  only  strains  H2  and  T9  produced 
leukocidins.  Thus  it  is  seen  that  H2,  which  did  not  show  hemolysin 
production  in  broth  cultures,  produces  most  leukocidin,  and  A5,  which 
produced  most  hemolysins,  does  not  produce  leukocidins.  Al  is  nega- 
tive in  both  cases,  while  T9  is  positive  in  both  cases.  However,  strains 
A5  and  H2  indicate  distinctly  that  hemolytic  and  leukocidin  activity 
are  not  dependent  on  each  other. 


26  L.    A.    JULIANELLE 

Theoretically  we  would  expect  that  the  amount  of  leukocidin  pro- 
duced would  bear  a  relation  to  the  virulence  of  a  strain,  for  the  latter 
would  depend  to  some  extent  on  the  former.  Since  virulence  and 
hemolysis  were  found  to  be  individual  characters,  it  was  hardly  supposed 
that  hemolysis  would  show  any  dependence  on  leukocidin  production. 

III.    SEROLOGIC    REACTIONS 

As  a  final  analysis,  recourse  was  taken  to  differentiate  the  hemolytic 
staphylococci  on  a  serologic  basis.  The  impression  is  that  although 
biochemical  reactions  may  vary,  serologic  reactions  if  once  positive  will 
always  remain  positive.  So,  for  example,  the  agglutinability  of  an 
organism  may  fluctuate  quantitatively,  but  not  qualitatively.  For  no 
other  reason,  then,  this  part  of  the  work  seemed  to  have  the  greatest 
promise.  Both  deviation  of  complement  and  agglutination  tests  were 
made,  and  the  agglutination  tests  were  supplemented  by  absorption  tests. 

In  preparing  immune  serums,  strains  Al,  A5,  PI,  T9  and  LI  were 
employed.  Salt  suspensions  were  made  from  agar  slants  and  rabbits 
were  injected  intravenously  in  3  day  periods,  with  2  days  between  each 
period.  Five-tenths  c  c  of  the  suspensions  was  injected  the  first  period, 
and  this  was  increased  0.5  c  c  each  period  until  a  serum  of  sufficiently 
high  titer  was  obtained. 

COMPLEMENT     FIXATION 

The  literature  on  the  complement  fixation  of  staphylococci  is  scant. 
The  one  reference  available  was  that  of  Kolmer,  Trist  and  Yagle  53  in 
relation  to  influenza.  Using  a  Staphylococcus  aureus  antigen,  they  were 
unable  to  get  fixation  with  either  normal  or  influenza  serum. 

The  antigens  used  in  these  experiments  were  suspensions  of  24 
cultures  to  which  were  added  0.1%  formaldehyd.  The  preparation  of 
the  serum  has  already  been  described. 

After  going  through  the  preliminaries  of  obtaining  antigenic  and 
complementary  doses,  the  tests  were  made  by  incubating  at  37  C.  It 
was  found  that  all  5  serums  gave  fixation  with  all  of  the  antigens. 
There  appears  to  be  no  qualitative  differentiation  of  the  different 
strains. 

One  more  step  was  taken,  and  that  was  to  determine  whether  there 
might  be  quantitative  separation  into  groups  by  complement  fixation. 
Four  strains  were  picked  at  random,  and  the  serum  used  in  dilutions 
of  1 :  50,  1 :  100,  1 : 150.  The  results  did  not  warrant  extending  the 

»  Jour.   Infect.  Dis.,   1919,   24.  p.    583. 


HEMOLYTIC  STAPHYLOCOCCI  27 

work  to  include  all  the  strains.  No  sharp  difference  in  the  ability  of 
the  strains  to  fix  complement  was  manifested,  as  the  serums  were 
increased  in  dilution. 

It  would  seem,  therefore,  that  staphylococci  are  able  to  fix  comple- 
ment in  more  or  less  the  same  degree.  Further,  the  reaction  is  a 
specific  one  for  antigens  prepared  of  streptococci  and  B.  friedlander 
were  unable  to  prevent  hemolysis.  But  no  evidence  is  given  of  a 
possible  classification  of  staphylococci  by  complement  fixation — either  in 
a  qualitative  or  quantitative  way. 

This  is  not  in  the  least  surprising,  however,  when  we  recall  that 
complement  fixation  does  not  show  divisions  into  groups  with  those 
cocci  which  have  been  proved  to  be  of  different  serologic  types  by 
agglutination  reactions. 

AGGLUTINATIONS 

The  agglutination  reactions  of  the  staphylococci  have  been  studied  by  sev- 
eral investigators.  Kolle  and  Otto  L'°  found  that  immunized  serum  distinguished 
the  pathogenic  from  the  nonpathogenic  forms.  This  was  confirmed  by  Klop- 
stock  and  Bockenheimer,53a  Van  Durme,3  Proscher,54  Kutscher  and  Konrich,22 
Veiel,55  Fraenkel  and  Baumann 58  and  Montegazza.49  Trincas  "  states  that  serum 
prepared  with  hemolytic  strains  shows  strong  agglutination  with  hemolytic 
strains,  and  slight  agglutination  with  nonhemolytic  strains ;  and  vice-versa. 
Walker  and  Adkinson  M  found  that  an  aureus  immune  serum  would  agglutinate 
aureus  and  not  albus  strains ;  and  that  an  albus  immune  serum  would  agglu- 
tinate albus  and  not  aureus  strains. 

Our  object  was  to  group  staphylococci  by  agglutination  into  as 
many  serologic  groups  as  would  evidence  themselves,  without  regard 
to  virulence  or  pigment.  The  same  serums  used  in  the  complement- 
fixation  test  were  used  for  agglutination,  and  the  same  antigens  also, 
except  that  they  were  diluted  until  their  turbidity  equaled  that  of  the 
Dreyer  standard  for  the  typhoid  group  agglutinations.  The  agglutina- 
tions were  set  up  in  serum  dilutions  of  1 :  10  and  going  as  far  as  was 
necessary  to  include  the  agglutination  titer  of  the  respective  serums. 
The  serum  dilutions  and  antigens  were  added  in  0.5  c  c  amounts  each, 
and  incubation  was  effected  in  a  water  bath  at  56  C.  for  16  hours. 

In  table  12  the  figures  represent  the  dilution  at  which  final  agglutina- 
tion was  observed  with  naked  eye.  There  was  present  in  the  serums  a 
proagglutinoid  zone. 

An  analysis  of  the  table  shows  that  serum  Al  agglutinates  strains 
Al,  Fl,  P2,  S2,  T2,  T5,  T8,  T9,  X,  CIS,  C16  and  CIS.  Serums  AS, 

5311  Centr.  f.   Bakt.,   1903,   34,   p.   437. 

54  Arch.  f.  klin.  Chir.,  1903,  72,  p.  325. 

55  Munchen.  med.   Wchnschr.,   1904,   51,  p.    13. 
M  Ibid.,    1905,  52,  p.   937. 

57  Biochem.   Centralbl.,   1908,  8,  p.   609. 

58  Jour.  Med.  Res.,   1917,  35,  p.  373. 


28 


L.    A.    JULIANELLE 


PI,  and  LI  agglutinate  strains  A2,  A3,  A5,  H2,  PI,  P3,  P4,  P5,  Tl, 
T2,  T3,  T6,  T9,  X,  Jl,  and  LI.  Serum  T9  agglutinates  P3,  T2,  T9 
and  X.  Serums  A5,  PI  and  LI  are  unquestionably  the  same  since 
they  give  the  same  reactions.  It  will  be  noted  that  strains  T2,  T9  and 
X  are  agglutinated  by  all  the  serums,  and  P3  by  all  the  serums  except 
Al.  Aside  from  these  atypical  agglutinations,  the  strains  fall  definitely 
with  one  serum.  Apparently,  then,  the  agglutination  tests  give  the 
following  grouping : 

L— Al,  Fl,  P2,  S2,  T5,  T8,  C15,  C16,  CIS. 

II.— A2,  A3,  AS,  H2,  PI,  P4,  P5,  Tl,  T3,  T6,  Jl,  LI. 

III.— T2,  T9  X  and  possibly  P3. 

TABLE     13 
RESULT  OF  ABSORPTION  TESTS  ANTIGENS  * 


Serum  Absorbed  With 


A1-A1 

Al-X 

T9-P3 

T9-T2 

L1-A5 

L1-P3 

L1-T9 

A  1... 

0 

4800 









A  2  





0 

300 

600 

A  3  





0 

300 

600 

A  5  







0 

300 

600 

F  1        

0 

+ 





H  2  



600 

300 

600 

P  1  





0 

300 

+ 

p  2  

0 

+ 





P  3  

0 

2400 

600 

0 

1200 

P  4  

0 

300 

+ 

p  5  





0 

300 

+ 

S  2  

0 

+ 





T  1  





0 

300 

+ 

T  2  

0 

0 

0 

300 

0 

0 

T  3  

0 

300 

+ 

T  5  

0 

+ 





T  6  





0 

300 

+ 

T  8  

0 

+ 







T  9  

o 

0 

0 

0 

300 

0 

0 

X  

600 

0 

0 

150 

600 

0 

0 

C  15  

0 

+ 





— 

— 

C  16  

0 

+ 











C  18  

0 

+ 









— 

J  1  





0 

300 

+ 

L  1  







0 

600 

1200 

*  —  indicates  that  strain  did  not   agglutinate  prior  to   absorption;   figures  represent  dilu- 
tion oi  final  positive  agglutination;   +  indicates  no  test.     All  controls  were  negative. 

ABSORPTION     TESTS 

In  order  to  further  identify  the  groups  suggested  by  the  aggluti- 
nation reactions  absorption  tests  were  conducted,  employing  the  technic 
of  Small  and  Dickson.59  One  c  c  of  a  1 :  10  dilution  of  the  immune 
serum  was  mixed  with  4  c  c  of  the  concentrated  antigen  in  a  sterile 
centrifuge  tube.  This  amount  of  the  antigens  was  found  sufficient  to 


69  Jour.   Infect.    Dis.,    1920,  26,   p.   230. 


HEMOLYTIC  STAPHYLOCOCCI  29 

absorb  the  homologous  agglutinins  after  4  hours'  incubation  at  37  C., 
the  tubes  being  shaken  at  half-hour  intervals.  After  this  period  of 
incubation  the  tubes  were  centrifugalized  and  the  supernatant  serum 
dilution  (1:50)  was  drawn  off  and  agglutinations  carried  out  as 
described. 

Serum  Al  was  absorbed  with  strain  Al  and  X ;  serum  T9  with  P3 
and  T2 ;  serum  LI  with  A5,  P3  and  T9,  and  agglutinations  performed 
against  the  antigens  which  agglutinated  with  the  respective  serum 
before  absorption.  The  results  are  presented  in  table  13. 

The  absorption  tests  confirm  the  groups  found  by  agglutination. 
Group  1  remains  as  was  found,  but  in  group  2,  H2,  is  placed  in  a 
subgroup  because  although  it  agglutinates  with  the  same  serums  as 
A5,  absorption  by  AS  does  not  remove  agglutinations  for  H2.  In 
group  3,  P3  is  placed  in  a  subgroup.  P3  removes  agglutinins  for  all 
members  of  group  3,  but  the  other  members  of  group  3  do  not  remove 
agglutinins  for  P3. 

Revising  our  classification,  then,  we  would  have : 

Group  3 
T2 
T9 
X 

Subgroup 
P3 


DISCUSSION     CF     SEROLOGIC     REACTIONS 

The  use  of  complement  fixation  in  determining  types  among  the 
staphylococci  appears  to  be  worthless.  Although  staphylococci  do  fix 
complement,  no  grouping  appeared  possible,  either  quantitatively  or 
qualitatively.  Nor  is  this  surprising — on  the  contrary,  it  is  more  or 
less  what  was  to  be  expected.  Complement  fixation  has  been  dis- 
appointing in  its  inability  to  differentiate  types — probably  because  the 
immunity  established  although  specific  for  the  particular  species  is 
general  and  not  sufficiently  specialized  to  detect  individual  types. 

Agglutination,  however,  has  already  been  proved  to  be  an  efficacious 
means  of  detecting  types.  Furthermore,  agglutination  is  a  fixed  quality, 
and  one  which  is  considered  reliable.  So  that,  when  the  statement  is 


30  L.    A.    JULIANELLE 

made  that  virulent  types  agglutinate  only  with  serums  prepared  from 
virulent  strains,  there  must  be  an  error  somewhere.  The  properties  of 
virulence  are  obviously  among  the  most  unstable  of  bacterial  characters. 
Culture  on  laboratory  mediums  renders  a  virulent  strain  nonpathogenic 
in  a  very  short  time.  Yet  it  is  scarcely  conceivable  that  the  immunity 
reactions  are  as  readily  modified.  By  way  of  illustration:  Strain  LI, 
which  was  distinctly  pathogenic,  was  used  for  the  preparation  of 
immune  serum  before  it  could  have  undergone  avirulence ;  but  its  serum 
did  agglutinate  other  strains,  including  A5,  P3  and  T9,  all  3  of  which 
were  proved  nonpathogenic.  It  may  be  that  A5,  P3  and  T9  were 
pathogenic  at  sortie  time  or  another,  but  at  the  time  the  test  was  made 
they  were  not  pathogenic.  It  seems  clear  to  us  that  virulence  does  not 
dictate  the  group  into  which  a  staphylococcus  shall  fall. 

Nor  does  it  seem  plausible  that  hemoly tic  activity  is  the  basis  of 
agglutination  grouping.  We  have  been  unable  to  obtain  absolutely  non- 
hemolytic  cultures,  and  have  been  unable  to  establish  this  point  con- 
clusively. However,  we  were  able  to  get  these  groups  among  hemolytic 
organisms,  whereas  if  hemoly  sis  were  the  fundamental  of  the  grouping, 
we  should  have  obtained  agglutination  of  all  our  strains  by  all  our 
serums. 

Regarding  the  association  of  pigment  and  agglutination,  this  much 
can  be  said :  Occasionally,  there  may  develop  on  a  plate  streaked  with 
a  pure  culture,  colonies  varying  appreciably  in  intensity  of  pigment, 
from  which,  as  Sullivan 60  has  shown,  quite  distinct  types  may  be 
derived  by  selection  of  the  extremes.  Yet  it  does  not  seem  probable 
that  the  parent  strain  in  such  a  case  would  vary  from  its  successor  in 
its  agglutination  reactions.  More  relevant,  however,  strain  Jl,  which  is 
an  albus,  did  agglutinate  with  serums  A5,  PI  and  LI,  which  were 
prepared  from  antigens  of  varying  shades  of  orange.  An  analysis  of 
the  pigment  and  agglutination  tables,  with  this  one  exception  cited, 
bears  out  the  contention  of  Walker  and  Adkinson  58  in  a  general  way. 
The  members  of  group  1  are  of  a  light  pigment — either  white  or  of  a 
light  shade  of  yellow,  which  without  the  refined  technic  of  Winslow  and 
Winslow  28  would  easily  be  called  a  white. 

A  study  of  the  tables  of  the  different  biochemical  reactions  shows 
no  definite  relationship  between  the  agglutination  groups  and  these 
reactions.  In  a  very  general  way  Group  1  seems  to  contain  the  less 
active  strains,  but  it  also  contains  some  rather  active  strains.  Groups  2 
and  3  possess  none  of  the  light  pigmented  nor  any  of  the  less  active 
strains. 

"»  Jour.   Med.   Res.,    irOS,    14,  p.   .09. 


HEMOLYTIC  STAPHYLOCOCCI  31 

These  immunologic  groups  will  perhaps  explain  the  variations 
experienced  in  curative  and  prophylactic  inoculations  of  either  the 
organisms  or  serum.  Stock  vaccines,  for  example,  will  not  necessarily 
be  specific,  nor  will  immune  serum  prove  to  be  efficacious  unless  it 
falls  into  the  same  group.  But  having  determined  the  group  or  type 
of  staphylococcus  under  question,  we  can  employ  specific  material  either 
prophylactically  or  curatively. 

CONCLUSIONS 

Staphylococci  produce  a  hemolytic  substance  in  broth  which  appears 
on  the  6th  day,  reaches  a  maximum  at  the  9th  or  10th  day  and  then 
disappears  between  the  13th  and  16th  day. 

This  hemolytic  substance  is  thermolabile,  is  unaffected  by  the 
presence  of  carbohydrates  and  appears  to  be  associated  with  proteolysis 
and  possibly  autolysis. 

All  cultures  of  Staphylococci  isolated  during  the  course  of  this 
investigation  appear  to  be  hemolytic — only  the  time  of  its  manifestation 
is  in  some  cases  considerably  delayed. 

Hemolytic  cultures  did  not  lose  their  hemolytic  powers  by  continued 
transplantations  into  blood-free  mediums  for  a  period  of  more  than 
four  months. 

Hemolytic  activity  shows  no  relationship  to  any  of  the  biochemical 
reactions  studied. 

Staphylococci  fix  complement  specifically,  but  cannot  be  classified 
by  such  an  expedient. 

Three  groups  seen  definable  of  the  25  strains  studied  by  agglutina- 
tions and  absorption  test,  with  2  ill-defined  subgroups — one  each  under 
group  4  and  group  3. 

These  groups  apparently  bear  no  relationship  to  virulent  hemolysis 
or  biochemical  activity.  Group  1  appears  to  include  the  light  pig- 
mented  and  less  active  strains. 

These  groups  may  account  for  the  variations  experienced  in  the 
past  in  the  use  of  serum  and  vaccines. 

The   writer   wishes   to    express   his   sincere   gratitude   to    Doctors   A.    C.    Abbott   and    D.    H. 
Bergey   for  their  invaluable  assistance   in    advice,   criticism  and  encouragement. 


9*486 


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